The present invention is directed to a mutein of Interleukin-6, its production by recombinant DNA technology, and its theraputic use.
Interleukin-6 (IL-6) is a multifunctional cytokine that is produced by a variety of cells such as B-cells, T-cells, monocytes, fibroblasts and endothelial cells. IL-6 exhibits several activities relating to the proliferation and/or differentiation of hematopoietic progenitor cells. These activities result from IL-6 acting alone or in combination with other cytokines such as IL-3 and IL-4. Some specific biological effects of IL-6 include terminal differentiation of B-cells, proliferation and differentiation of T-cells, regulation of the acute phase response, growth regulation of epithelial cells, the differentiation of megakaryocytes, and thrombopoiesis. In accordance with these activities and effects, the target cells for IL-6 include B-cells, T-cells, myeloma cells, megakaryocytes, monocytes, early stem cells and hepatocytes.
Though IL-6 is a multifunctional cytokine, the various biological effects it exerts are believed to be initiated by the stepwise interaction of IL-6 with two distinct receptor subunits on a cell. IL-6 first forms a complex with an 80 kD receptor subunit. This complex binds to a non-ligand subunit, which is a membrane glycoprotein designated gp130. The binding of the IL-6-80kD receptor complex to gp130 results in signal transduction.
The amino acid sequence of IL-6 has been described in the literature as containing 185 amino acids starting with alanine as residue 1; see, for example, FIG. 2A of Brakenhoff et al., J. Immunol. 139, 4116-4121 (1987) and FIG. 1 of Clark et al., PCT application WO 88/00206. These references also contain the cDNA sequence that corresponds to native IL-6 mRNA.
The carboxy terminus of IL-6 has been found to be the receptor binding region. (See Leebeek, F. W. G., et al., "Identification of a Receptor Binding Site in the Carboxyl Terminus of Human Interleukin-6", Journal of Biological Chemistry, 267, 14832-14838 (1992) Analysis of the structure of the carboxy terminal region of several cytokines, including IL-6, has shown the binding region to be highly conserved. Lokker, N., et al., EMBO J., 10, (8) 2125-2131 (1991). See page 2130, top of col. 1.; BAZAN, Immunology Today 11, 350-354 (1990). According to Lokker et al., "[S]ubstitutions in the . . . C-terminal proximal regions of these molecules abolished their activity."
New techniques in molecular biology, such as recombinant DNA and monoclonal antibodies, make it possible not only to study the relationships between the structure and function of proteins, but also to enable the production of protein analogs with superior properties. The analogs may differ from native proteins by deletion, substitution, or addition of amino acids to the native sequence. Since the DNA and amino acid sequences of IL-6 are known, these techniques can be used to study and improve the various properties of IL-6.
For example, the deletion of amino acids may give important information about the function of a protein. Thus, Brakenhoff et al. have reported that the biological activity of IL-6 is not affected by deletion of up to 28 amino acid residues from the amino terminus (N-terminus) of mature, native IL-6. Brakenhoff, J. P. J., et al., J. Immunol. 43, 1175-1182 (1989).
Conversely, deletion of only a few amino acids from the carboxy terminus (C-terminus) of IL-6 has a pronounced effect on biological activity. For example, Kruttgen et al. found that a deletion of the last amino acid from the carboxy terminus of IL-6 resulted in a fivefold loss of biological activity. Furthermore, IL-6 lacking three or four carboxy terminal amino acids was found to be completely inactive. Kruttgen, A-, et al., FEBS Lett. 273, 95-98 (1990).
Since these truncated forms of IL-6 were not tested for binding to the IL-6 receptor, it remains unclear whether the loss of activity observed was due to a loss of receptor binding or to a defective signal transduction upon IL-6 receptor interaction. In any event, the importance of the presence of the C-terminal amino acid residues is consistent with the highly conserved nature of this region, see above.
Additional information about the importance of the carboxy terminus of IL-6 for biological activity was obtained by Brakenhoff et al. These authors showed by epitope mapping that neutralizing monoclonal antibodies against IL-6 were directed to a region of the carboxy terminus. Brakenhoff, J. P. J., et al., J. Immunol. 145, 561-568 (1990).
Native proteins may be improved by substituting amino acids for one or more of the amino acids that occur naturally in a protein. Such substitutions may be introduced into a protein by expressing recombinant DNA having a nucleotide sequence modified so as to have a codon that represents the desired amino acid. Proteins expressed by such modified DNA are called muteins. The DNA may conveniently be modified using the technique of saturation mutagenesis. Hutchinson, C. A., et al., Proc. Natl. Acad. Sci., U.S.A. 83, 710-714 (1986).
In a typical example of a mutein, native cysteine residues are replaced by other amino acid residues. Substituting other residues for cysteine residues may make it easier to express proteins in bacteria. Snouwaert, J., et al., J. Immunol. 146, 585-591 (1991); See page 589, col. 1, last paragraph bridging col 2, through first full paragraph.
Muteins of IL-6 with improved biological activity relative to native IL-6 are desirable. An IL-6 mutein with enhanced biological activity would permit a lower dosage of the mutein to be administered to patients. Therefore, the costs of treatment and manufacture would be decreased with the more active IL-6 mutein. In addition, a lower dosage of the more active IL-6 mutein may reduce or eliminate possible side effects of the medication.
It is the principal objective of the present invention to produce IL-6 muteins that have amino acid substitutions such that an increase in biological activity is achieved.
Other objectives are to produce DNA, vectors and plasmids that encode muteins of IL-6 with increased activity.